Int. J. Nanotechnology, Vol. x, No. x, xxxx 1

Copyright © 200x Inderscience Enterprises Ltd.

Biomimetic membranes and biomolecule immobilisation strategies for nanobiotechnology applications

Agnes P. Girard-Egrot, Christophe A. Marquette and Loïc J. Blum * Laboratoire de Génie Enzymatique et Biomoléculaire Institut de Chimie et Biochimie Moléculaires et Supramoléculaires ICBMS; UMR5246; Université Lyon1 – CNRS 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France Fax: +334 72 44 79 70 E-mail: Agnes.Egrot@univ-lyon1.fr; christophe.marquette@univ-lyon1.fr; Loic.Blum@univ-lyon1.fr *Corresponding author

Abstract: Biological membranes constitute a source of inspiration for making supramolecular assemblies which can be used in the design of biomimetic sensors. At the same time, the concept of using biomolecules as an elementary structure to develop self-assembled entities has received considerable attention. More particularly, the ability of amphiphilic molecules like lipids, to spontaneously organize into bilayers, is suitable to achieve biomimetic membrane models. The potential of two-dimensional molecular self-assemblies is clearly illustrated by Langmuir monolayers of lipids formed at an air/water interface, which can be used as models to acquire knowledge about the molecular recognition process occurring in biological membranes. Langmuir-Blodgett (LB) technology, based on the transfer of this interfacial monomolecular film onto a solid support, allows building up lamellar lipid stack, with an accurate control of thickness and molecular organisation. This technique offers the possibility to prepare ultrathin layers suitable for biomolecule immobilization. We are presenting herein an overview of work performed in our group that sheds light on the formation of biomimetic LB membranes associating protein in a well-defined orientation. Two points will be addressed: investigations of protein/lipid interactions using lipid monolayers as membrane models and biosensing applications. The objectives are to highlight advantages of interfacial Langmuir monolayers and supported Langmuir-Blodgett films to investigate molecular interactions between biomolecules and lipid membrane components or to elaborate biomimetic membranes as sensing layers, respectively. The present article also draws a general picture of non-conventional methods for biomolecule immobilization and their applications for biochip developments. The technologies presented are based either on original solid supports or on innovative immobilization processes. First, “Macromolecules to PDMS transfer” technique relying on the direct entrapment of macromolecules spots during PDMS polymerisation is proposed as an alternative for the easy and simple PDMS surface modification. Then, the electro-addressing of biomolecule-aryl diazonium adducts at the surface of conducting biochips will be presented and shown to be an interesting alternative to immobilization

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processes based on surface functionalization

Keywords: Biochip; Biomimetic membrane; Microarray; Langmuir-Blodgett film; Lipid bilayer;

Reference for publisher use only

Biographical notes:

Prof. A.P. Girard-Egrot received the Doctorat de spécialité in Biochemistry (1995) and the “Habilitation à Diriger des Recherches” (2002) from Université Claude Bernard-Lyon 1. She is presently Professor of Biochemistry at the same university and is in charge of development of biomimetic membranes based on Langmuir-Blodgett technology. Since 1993 author or co-author of more than 25 articles and book chapters. Dr. C.A. Marquette received the Doctorat de spécialité in Biochemistry (1999) from the Université Claude Bernard-Lyon 1. He is presently permanent researcher at the Centre National de la Recherche Scientifique (CNRS) at the UCBL and is in charge of the development of optical biochips and micro-arrays based on luminescent reactions. Since 1998 author or co-author of more than 50 articles and book chapters. Prof. L.J. Blum received the Doctorat d’Etat ès Sciences (1991) from the Université Claude Bernard-Lyon 1. He is presently Professor of Biochemistry and Biotechnology at the same University and is involved in the development of biosensors, bioanalytical micro and nano systems and biomimetic membranes. He is the head of the UCBL/ CNRS research unit ICBMS. Since 1983 author or co-author of 150 articles and book chapters.

1 Biomimetic membranes for nanobiotechnology applications Biological membranes play a central role in the cell life. Besides their compartment-talization function, they are involved in many exchange processes between the outside and inside cellular worlds. Only a few manometers thick, biological membranes consisted of two main components, have a perfect organization on the molecular level. Lipids, held together by hydrophobic interactions, essentially play the structural role, forming a continuous bilayer acting as a diffusion barrier. Proteins, like transmembrane proteins or peripheral membrane proteins, respectively embedded within the membrane or transiently associated with it, are devoted to either exchange or biocatalysis processes. Consequently, biological membranes, highly complex and dynamic supramolecular structures, are a key component of the way that living cells are able to maintain and organize their function. Unlocking the secrets of those membranes provides important lessons that are valuable in guiding the construction of devices to be used for nanotechnological applications [1]. In particular, molecular and supramolecular understanding of the architecture of biological membranes constitutes an extraordinary source of inspiration for making ‘intelligent’ nanostructures which can be used in the design of biomimetic sensors based on the molecular recognition and signal transduction events occurring in natural membranes. Nowadays, direct contact between nanostructures mimicking cell membranes and electronic devices offers a direct way of

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following molecular processes (or biocatalytic reactions) by studying a very limited number of molecules. Actually, bioelectronic interfacing between living and inert matter lies at the heart of nanobiosciences. In this sense, biomimetic membranes provide basic support structure for many applications in nanobiotechnology. At the same time, access to the complex functioning of biological membranes is a real challenge. Exploratory studies are performed both on integrated and on reconstituted systems using models of natural membranes. Hence, since the complexity of cell membranes, reliable models to acquire current knowledge of the molecular processes occurring at biological membranes, either for studying basic membrane processes or for technological applications need to be achieved. Natural membrane organization builds upon the self-association properties of biological molecules making them up. In vitro, these provide the basis for a natural and spontaneous formation of bilayer structures which can be exploited to reconstitute biomimetic membranes and a wide range of protein-lipid nanostructures. Hence, the concept of using biomolecules as an elementary structure to develop self-assembled entities corresponding to organized supramolecular arrangements has thus received considerable attention [2]. More particularly, the self-assembly ability of amphiphilic biomolecules such as lipids, to spontaneously organize into nanostructures mimicking living cell membranes, has appeared as a suitable concept for the development of biomimetic membrane models [3-5]. At the same time, the growing interest in confining lipid membranes on solid support has been nourished by the emergence of a wide range of surface-sensitive characterization techniques which can be applied to study model characteristics or proteins/membrane interactions. The potential of two-dimensional molecular self-assemblies is clearly illustrated by Langmuir monolayers of lipid molecules formed at an air/water interface, which can be used as models to understand the role and the organization of biological membranes [6], or to acquire knowledge about the molecular recognition process [7-9]. Langmuir-Blodgett (LB) technology, based on the transfer of this interfacial monomolecular film onto a solid support, allows building up lamellar lipid stacks. When all transfer parameters are optimized, this technique corresponds to one of the most promising for preparing supported lipid membranes as it enables (i) an accurate control of the thickness and of the molecular organization, (ii) an homogeneous deposition of the monolayer over large areas compared to the dimension of the molecules, (iii) the possibility to transfer monolayers on almost any kind of solid substrate and (iv) to elaborate bilayer structures with varying layer compositions. Based on the self-assembled properties of amphiphile biomolecules at the air/water interface, LB technology offers the possibility to prepare ultrathin layers suitable for immobilization of bioactive molecules. Here we present an overview of work performed in our group that sheds light on the formation of biomimetic LB membranes associating protein in a well-defined orientation. Two points will be addressed: biosensing applications and investigations of protein/lipid interactions using lipid monolayers as membrane models. The objectives are to highlight advantages of interfacial Langmuir monolayers and supported Langmuir-Blodgett films to investigate molecular interactions between biomolecules and lipid membrane components or to elaborate biomimetic membranes as sensing layers, respectively.

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1.1 Langmuir monolayer formation and Langmuir-Blodgett technology Langmuir monolayer technique is based on the properties of amphiphile biomolecules like lipids to orient themselves at an air/water interface and to form an insoluble monolayer called Langmuir film (Figure 1). When amphiphile molecules are deposited on the surface of water, the dispersion forces quickly cause the solution to spread over the whole available surface. After solvent evaporation, lipid headgroups are immersed in the subphase, the hydrophobic tails pointing toward the gaseous phase. The interfacial film resulting is a monomolecular layer of one-molecule thick.

The monolayer, initially present in a gaseous phase, is then compressed by two mobile barriers. As the available surface area of the monolayer is reduced, the molecules start to interact and the surface tension lowered. During compression, the molecules self-assembled at the interface to form a homogeneous interfacial film. The condensation state of the molecules is directly correlated to the surface pressure (π) increase recorded by a Wilhelmy plate partially immersed through the interface. The surface pressure (π) corresponding to the force exerted by the film per unit length equals:

π = γ0 - γ (1)

where γ0 is the surface tension of the pure liquid and γ the surface tension in the presence of a monolayer. During this process, the hydrophilic and hydrophobic ends of the molecule ensure that the individual molecules are aligned in the same way.

Figure 1 Langmuir monolayer formation

1. Molecules spread at the air/water interface

Formation of a monomolecular film in a gaseous state

2. Monolayer compression by two mobile barriers

Molecular self-organisation at the interface and formation of an interfacial film in

different aggregation states

3. Monolayer in condensed state

Monomolecular film perfectly organised at the air-water interface at the end of compression

π

Surface pressure (π)measurement

LB trough

Wilhelmy plate(tensiometer)

When the surface pressure is sufficiently high to ensure lateral cohesion in the interfacial film, the floating monolayer can be transferred, like a carpet, from the water surface onto a solid support (Figure 2). During the transfer, the surface pressure is maintained constant by a feedback servoloop of the compression system. In the Langmuir-Blodgett technology [10, 11], the film deposition arises from the vertical movement of a solid substrate through the monolayer/air interface. Depending on

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whether the substrate is hydrophilic or hydrophobic, the first monolayer will be transferred as the substrate is respectively raised or lowered through the interfacial film. Subsequently, bi- or multi-layer stacks, called Langmuir-Blodgett films, are produced by deposition of one monolayer each time the substrate goes through the interface. Hence, the lamellar arrangement is representative of the natural lamellar stack of the biological membrane.

The LB technique offers the possibility to perfectly control each step of LB films formation. The main advantage with LB membranes lies in the highly ordered molecular arrangement, achieved on the water surface and conserved during transfer onto the substrate when all transfer parameters (surface pressure, rate of immersion of the substrate, temperature, composition of the aqueous phase) have been optimized [12-14]. Akin to the biological bilayer, the structure of LB films makes them candidate for developing biomimetic models of natural membranes.

Figure 2 Langmuir-Blodgett deposition

Vertical transfer onto a solid support

Lamellar arrangement

Hydrophobic supportHydrophilic support

Constant surface pressure

1.2 Investigations of molecular interactions in Langmuir monolayers as membrane model

As previously shown, Langmuir monolayers are monomolecular films formed at the air/water interface. Due to their organization similar to the leaflet of the natural phopholipidic bilayer, phospholipid monolayers represent in turn an attractive membrane model, the thermodynamic relationship between monolayer and bilayer membranes being direct [15].

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Figure 3 Langmuir monolayer as membrane model

The main advantages of this model are the achievement of a molecular state perfectly organized at the water surface and the control of the aggregation state by the lateral pressure imposed to the lipids in well-defined physicochemical conditions (subphase composition, pH, ionic strength, temperature) [16]. Mainly, monolayers overcome the limitation to regulate lipid lateral-packing density and lipid composition independently, as encountered for liposome dispersions. Especially, all the molecules forming the monolayer are in the same orientation (absence of curvature or constraints at the level of the phospholipid polar groups). Hence, Langmuir monolayers have been extensively used for studying membrane interactions of peptides [17-19] or proteins [9]. Generally speaking, Langmuir monolayers may be used to characterize the penetration capacity of biomolecules in a lipid membrane at a molecular scale. They are also informative on the stability of the molecule in the lipid environment.

Figure 4 Principles of molecular interaction investigations between biomolecules and Langmuir monolayer

Surface pressure (π)measurement

Biomimetic membrane modelBiological membrane leaflet

Surface Pressure (π)

Time

Injection

Molecule injection under monolayer at constant surface area

Surface pressure (π)

π

Time

Affinity of the protein for the monolayer

(∂π/∂t)t=0

Exclusion surface pressure :“penetration capacity”

π initial

∆π

Initial velocity of surface pressure increase Extrapolation of the linear

plot at ∆π = 0

Analysis of penetration kinetics

∆π

Surface Pressure (π)

π initial

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Langmuir monolayer allows the investigation of molecular interactions between biomolecules and membrane components forming the membrane leaflet when a molecule is injected in the aqueous phase under the monolayer compressed at a defined surface pressure (π initial). If it inserts itself into the interfacial film, thus indicating an interaction, the surface pressure increases, provided that the area is held constant (Figure 4). This topography allows simulating, under realistic biological conditions, what happens when hydrosoluble molecules (peptides, cytoplasmic proteins, hormones, probes, etc.) interact at the surface of target cell (or organelle) membranes. The molecular interactions with the interfacial film lead to a time-dependent surface pressure increase (when the monolayer surface area is fixed). The analysis of the penetration kinetics allows getting insights in both the penetration capacity and the affinity of the molecule for the lipid constituting the monolayer. Moreover, the variation of the surface pressure in the interfacial film after insertion is an indicator of the stability of molecules in the lipid environment. At the same time, the surface morphology of the film inserting the molecule may be observed by Brewster Angle Microscopy (BAM) [20-23]. BAM is a powerful in situ surface investigation technique which allows a direct visualization of the lipid domain morphology formed in the monolayer at the air/water interface. It gives some information about the homogeneity of the interfacial film. This technique is informative on the structural rearrangement which can occur during interaction of molecules with lipid monolayer. In this context, BAM may be useful to know if the interacting molecule can modify or not lipid membrane organization.

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Figure 5 Morphology of mixed PC:PE (2:1) monolayers in interaction with CRMP5. BAM image shows the morphology of the proteo-lipidic monolayer at the air /buffer interface. AFM images were taken after transfer of the monolayer onto a silica substrate at a surface pressure of 21 mN/m. Height differences confirms exclusion of CRMP5 from the condensed domains and shows that it mainly concentrates at the periphery of condensed domains.

For instance, we have recently investigated in our group, the interaction of CRMP5 [24], a member of collapsing response mediator protein (CRMP) family, with phospholipid Langmuir monolayer as model to estimate its insertion ability. The collapsin response mediator proteins are strongly expressed in the developing brain where they take part in several aspects of neuronal differentiation. Their expression is down-regulated in the adult brain, but, CRMPs are expressed again with high levels during some cancers or neurodegenerative diseases. Among them, the role and the biochemical functions of the most recently discovered CRMP5 remain obscure. While present in cytosol, CRMP5 has been also localized in some cell membrane fractions. Using Langmuir monolayers, we have showed that CRMP5 is able to penetrate into both monolayers composed of PC1 or PE2, used as phospholipids representative of the biological membranes (data not shown). With a mixed monolayer composed of PC and PE at a 2:1 molecular ratio, BAM and AFM techniques demonstrate that CRMP5 localizes preferentially in the fluid phase of the monolayer with a high tendency to exclude from the condensed phase (Figure 5). This study confirms CRMP5 potentiality for interacting with cell membranes and illustrates

1 Phosphatidylcholine 2 Phosphatidylethanolamine

AFM images

Condensed lipid domainsof ≈3 nm height

5 µm

10 µm

2 µm

50 μm

21 mN/m

350 µm

Mixed proteo-lipid domainsof ≈6 nm height

BAM image

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the potentiality of the Langmuir monolayer as membrane model to investigate lipid/protein interactions or macromolecules in general.

1.3 Functionalised Langmuir-Blodgett lipid films as supported biomimetic membrane models: applications in Nanobiotechnology

Lipid membranes are self-assembled entities that can be used in a general manner as substrates for the immobilization of biomolecules which may have specific biological activities. The functionalisation of lipid membranes to develop protein–lipid assemblies is a crucial step in many applications in nanobiotechnology. The functionalisation of Langmuir-Blodgett lipid films can be achieved by association of proteins presenting specific recognition properties, such as enzymes, antibodies, receptors or specific ligands, in order to develop ordered protein–lipid molecular assemblies. These supported biomimetic membranes, corresponding to supramolecular arrangements, can be used to functionalise surfaces, upon which the protein confers its biospecificity. Over the past twenty years, a lot of research has been carried out on the association of proteins, and in particular enzymes, with Langmuir–Blodgett films. The bioactive films obtained in this way have been studied for their potential applications in the design of biosensors, with the protein–lipid LB membranes integrated into these systems as ultrathin sensitive films [25]. Since they can be transferred on various types of substrate, these films exhibit many advantages for the development of novel micro- or nanobiosensors, inspired by biological models. Especially, biomimetic biosensors based on the direct transduction of biological signals like in the biological membrane can be produced. As for other systems mimicking biological membranes, their structural organisation (highly ordered) and their ultrathin dimensions (a few nanometres thick) are the main characteristics for designing micronic sensors operating on the molecular scale and displaying ultra-rapid response times, fundamental criteria for further development of ‘smart’ sensors or biochips. However, the interest of LB films is not limited to these structural aspects. Specific advantages are worth mentioning: (i) the elaboration of a bioactive sensing layer and its association with the transducer is achieved in a one-step procedure, (ii) only a very small amount of protein is required to prepare the membrane, (iii) experiments are performed at ambient pressure and temperature hence avoiding the kind of thermal treatments required in the design of electronic systems, which would damage biological components, (iv) the performance of the sensor in terms of detection limit, sensitivity and dynamic range can be modulated by varying the number of the deposited protein–lipid layers [26-29].

The crucial stage in the fabrication of supported biomimetic LB membranes remains the incorporation of the biological element in LB films, without alteration or loss of activity. Several methods have so far been developed to produce active self-associated protein–lipid assemblies in bilayers or multilayers [25]. The most commonly used derives from the procedure developed to study protein–lipid interactions with a Langmuir monolayer [15, 30, 31]. It corresponds to the adsorption of the protein present in the subphase onto the interfacial film before transfer of the mixed proteo-lipidic monolayer [27, 30, 32-43]. This method is particularly well suited to extrinsic peripheral proteins capable of associating with biological membranes or to anchoring proteins inserting themselves into one leaflet of a bilayer. However, it presents some drawbacks [44]. The presence of the

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protein in the interfacial film may affect its transferability properties [26, 30, 45]. The surface pressure required for the transfer procedure may not be always well suited for the enzyme association (ejection of the protein at high surface pressure). The presence of the protein may induce a poor monolayer adhesion on the substrate, leading to a peeling-off at the subsequent immersion. Another approach consists in adsorbing the protein onto pre-formed LB film [46-50]. The main advantage with this procedure lies on the possibility of associating the protein with a hydrophilic lipid surface (polar head at the surface) or a hydrophobic lipid surface (hydrocarbon chains at the surface), depending on the number of layers deposited on the substrate. Nevertheless, the interactions involved in this type of association are often too weak to prevent the release of protein molecules which remains a major drawback as previously shown in our group [51] or reported elsewhere [52], and is often the main reason explaining the poor reproducibility of responses of LB membrane-based sensors. In order to minimise desorption of protein molecules, some authors have suggested to covalently immobilise the protein on LB film surfaces by means of cross-linking agents [53, 54]. The stabilisation of the proteo-lipidic LB films by reticulation after transfer with glutaraldehyde vapour has been also investigated [45, 55, 56]. The fact remains, however, that covalent attachment to the lipid structure may induce changes in the protein conformation, which may cause a loss of its biological activity. Another alternative for limiting desorption and avoiding covalent immobilisation of the protein has been proposed in our group. It consists in covering the protein molecules by transferring a further lipid layer onto the surface of the adsorbed molecules [46, 57-59]. This procedure referred to as “inclusion process” allows the sandwiching of the enzyme in a hydrophobic or a hydrophilic environment while keeping the homogeneity of the supporting layers. The specific features of these latter methods are the possibility to easily modify the lipid composition of the protective leaflet [60] and, to some extent, to reproduce the membrane asymmetry which can favour the physical retention of the protein and preserve its biological activity.

he association of proteins, and especially enzymes, with Langmuir–Blodgett films, using the techniques presented above, has recently been reviewed in [25], which discusses in particular the main points of interest of such biomimetic membranes and its applications in nanobioscience.

1.4 Biomimetic LB membrane inserting oriented proteins

The functionalisation of Langmuir–Blodgett films by association of proteins before or after transferring the lipid leads to a random association of the protein to the biomimetic LB membrane. One of the great challenges in the development of ordered protein–lipid assemblies and functionalised biomimetic membranes is to control the orientation of the associated protein, just as it is in biological membranes where the binding of the protein on (or in) the lipidic leaflets determines its own orientation for an optimal functionality. The building-up of organized proteo-lipidic membranes possessing properly oriented recognition sites constitutes a promising model for further developments in biomimetic sensing layers. It is of great interest in nanobiosciences and nanobiotechnology for many reasons. These reasons include, (i) used in contact with a chemical (or physical) device handling as a signal transducer, such membrane models may open a new way in the biocatalysis investigations to access the complex functioning of biological membranes at

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a nanometric scale; (ii) associated to microelectronic and optoelectronic devices, they should lead to the design of new bioelectronic hybrids and the development of novel nanobiosensors based on molecular recognition and signal transduction events of biological systems; (iii) deposited on an ovoid scaffold and integrating ion channels or pore proteins, they may be implied in the drug vectorisation and drug delivery [61]. In order to overcome the problem of the orientation of the protein associated with lipid membranes in general, and Langmuir–Blodgett films in particular, several strategies have been developed independently. These include the covalent coupling of the antigen binding fragment of an antibody via a disulfide bridge to the polar headgroup of a linker lipid inserted into a phospholipid monolayer [62-64], or the immobilisation of histidine-containing proteins onto metal ion chelating lipid monolayers [65]. However, in this way, several orientations, defined by the spatial distribution of the histidine residues on the surface of the protein, may be obtained. The possibility of immobilising glycosylphosphatidylinositol (GPI)-anchored proteins may circumvent the problem of multiple orientations. The unique orientation of the protein is then guaranteed by inserting its anchor into the phospholipidic LB films [66]. However, although this method is as biomimetic as one could hope, it only works for a well-defined class of proteins.

With the aim of designing functionalised biomimetic membranes with unique orientation of recognition sites, another strategy has been recently developed. The idea is to insert a monoclonal antibody that does not inhibit biological activity in a Langmuir–Blodgett lipid bilayer. The antibody serves as an anchor to tether the protein in an oriented position at the membrane surface (Figure 6), both to avoid denaturation of adsorbed protein onto lipid surfaces and to preserve biological activity over few months [67]. The membranes obtained are polyvalent and the nature of the protein that is retained is defined by the specificity of the inserted antibody. In this original approach developed in our group, the functional insertion of the antibody in the lipid membrane has been achieved by using a suitable combination of two techniques based on molecular self-assembling properties: liposome fusion at an air-buffer interface and Langmuir-Blodgett technology. This procedure exploits the possibility of forming a mixed monomolecular proteo-lipidic film at the air/buffer interface using surface tension forces able to disrupt membranes of a weakly stable protein–lipid vesicle [68, 69]. After compression, the mixed monolayer is transferred by LB transfer [70]. The principal interest about preparing protein–lipid vesicles before forming the interfacial monolayer is that interactions can then be set up by self-association between the lipid molecules and the antibodies in the vesicle membranes to improve insertion of the antibody in the interfacial film and hence, transfer the film without ejection of the protein. The vesicles are thus used as vectors for carrying the antibody directly to the air/buffer interface in a lipid environment [71].

Hence, by combining these two techniques, i.e., liposomes and the LB technique, the orientation of the antibody in the liposome membrane can be predetermined, and this orientation will be preserved when the liposomes open at the interface. The specific interactions initially formed in the vesicle membrane are preserved during the interfacial vesicle disintegration and lead in turn to a preferential orientation of the antibodies in the supported bilayer structure [72]. The organisation of the protein–lipid film is then maintained by lateral compression of the monolayer. After immunoassociation, the target

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protein will be retained at the surface of the bilayer membrane in a well-defined orientation [67].

Figure 6 Structural model of functionalised LB membrane dedicated to the oriented immobilisation of proteins. The lipid bilayer is made from a synthetic neoglycolipid presenting highly-fluid hydrocarbon chains. The monoclonal antibody is held in the bilayer by (i) assumed carbohydrate interactions between the glycan moiety of the antibody and the polar headgroups of the glycolipid, and (ii) hydrophobic interactions between the Fc fragment of the antibody (a region rich in aliphatic residues) and the lipid moiety of the glycolipids leaflet. In this model, the acetylcholinesterase enzyme (AChE) is associated to the functionalised lipid bilayer after LB transfer on a solid support, by specific immuno-recognition of the non-inhibitor antibody. This biomimetic membrane is structurally stable and can maintain AChE activity over several months. From Godoy et al [72].

AChE enzyme

Antibody

7 nm

5 nm

10 nm

Synthetic neoglycolipid

1.5 Potential applications of supported biomimetic LB membrane associating protein in a well-defined orientation

From a structural point of view, the main characteristic of biomimetic membranes associating protein in a well-defined orientation is their great stability. These functionalised biomimetic membrane remain stable and functional for several months. After immunoassociation of enzyme as protein model (i.e. acetylcholinesterase (AChE) involved in the neurotransmission of the nerve influx and target of many environmental pollutants like organophosphorus agents, Figure 6), the membrane stability allows to retain efficient enzyme activity for a long period of time (over a period of 82 days) [67]. To our knowledge, such a high stability has never been reported previously for an immobilised enzyme onto Langmuir-Blodgett films.

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1.5.1 Biocatalysis investigations

Thank to such a high functional stability, which is a prerequisite for relevant investigations, theses membranes can be used to investigate the kinetic behaviour of an enzyme in a lipid environment on a few nanometres thick membrane model. The results obtained with acetylcholinesterase as enzymatic model, clearly demonstrated a catalytic enzyme behaviour characteristic of immobilised enzymes, with the marked effects of diffusion constraints (for high substrate concentrations) in the microenvironment of the enzyme [73]. Hence, these functional biomimetic membranes, possessing properly oriented recognition sites by allowing an oriented binding of the enzyme at the surface of the lipid bilayer and offering a similar topography to the one found in biological membranes, represent a great potential for biocatalysis investigations in a general manner, and for fundamental assessment in the field of enzymology in structured media in particular. Most enzymes in cells are held in membranes and are found in a phospholipid environment that is absolutely necessary for them to function correctly. The catalytic behaviour of enzymes immobilised on a lipid bilayer is in turn fully representative of enzyme biocatalysis of the kind observed in vivo.

1.5.2 Design of bio-optoelectronic micro/nanosensors

Biomimetic membrane associating protein in a well-defined orientation can be used to functionalise micronic surfaces by integrating biochemical functions and to develop biomimetic sensors exploiting sensitive layers structured on the nanoscale. Biochemical sensors, or biosensors for short, are high-performance analytical tools, combining the specific recognition capacity of a sensitive biological element, the bioreceptor, with the sensitivity of the (electro-)chemical, physical, or optical sensor, the transducer. The latter detects physicochemical changes generated by the bioreceptor upon contact with the target substance and translates them into a measurable and interpretable electrical signal. The performance of a biosensor is closely linked to the properties of the sensitive layer and the quality of its association with the transducer. Current developments follow the marked trend toward miniaturisation of recognition systems. Molecular scale patterning of the sensitive layer is therefore a crucial step in sensors miniaturisation.

With the aim of designing a new miniaturisable bio-optoelectronic sensor, biomimetic membranes obtained by the Langmuir–Blodgett technique and associating aacetylcholinesterase (AChE) in an oriented way has been combined with a high-performance optical sensor (Figure 7) [73]. This new type of sensor combines the advantages of using a biomimetic membrane as sensitive layer with those of using screen-printed electrodes for the electrochemi-luminescence reaction of luminol (ECL): (i) the membrane is ordered at the molecular level, so there is hope for miniaturising the analysed region, (ii) it allows a functional orientation of the enzyme at the membrane surface, something that is not always possible with the usual methods for immobilising enzymes, (iii) the membrane, exploiting the specific recognition properties of a non-inhibitor monoclonal antibody, is multipurpose, whence different enzymes could be immobilised there by changing the antibody.

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At the same time, since the detection system is triggered by hydrogen peroxide, it can be applied to many oxidases able to detect a range of different metabolites of medical, industrial, or pharmaceutical interest. Environmental applications are also envisaged. Finally, the intimate contact between the different enzyme layers favours the flow of metabolites toward the detection device, avoiding back-diffusion into the reaction medium and increasing sensitivity. The ultrathin dimensions of the biomimetic membrane lead to the high performance of this sensor, especially in terms of response time.

Figure 7 Bio-optoelectronic microsensor based on biomimetic LB membrane. This sensor has been obtained by direct transfer of a biomimetic Langmuir–Blodgett membrane associating acetylcholinesterase (AChE) in oriented position at the surface of a screen-printed electrode adapted for electrochemiluminescence reaction of luminol. Briefly, acetylcholinesterase catalyses choline formation from acetylcholine present in the reaction medium. Choline is then oxidised by choline oxidase (ChOD) immobilised in a photopolymer of poly(vinyl alcohol) (PVA) at the surface of a screen-printed electrode. This produces hydrogen peroxide (H2O2), which is detected in the presence of electro-oxidised luminol by light emission focused on an optical fibre connected to the photomultiplier tube of a light meter. From Godoy et al. [73].

Working area(0.18 cm2)

graphite Biomimetic LB membrane(Neoglycolipid-antibody-AChE)

ca 10 - 20 µm 17 nm

+ 450 mV vs Ag/AgCl

+ hν

λmax. = 425 nm

FiberopticLuminol

diazaquinone

H2O2

Choline sensor

acetylcholinecholine

Screen-printed microelectrode

Choline oxidase (ChOD) included in PVA polymer membrane

However, as it is designed, the performance of this sensor also depends on the additional introduction of luminol during ECL measurement. Injections of soluble luminol in the reaction medium inevitably result to a lag-time due to diffusion of this reactant through the sensing layer to trigger ECL reaction. The possibility to insert an amphiphilic luminol derivative directly into the lipid bilayer as support for ECL detection may give the opportunity to develop reagentless biomimetic sensors (Figure 8). This amphiphile derivative has been recently synthesized in our group. Its potential activity for ECL measurement has been first investigated [74]. Its interfacial behavior and its ability to form stable monolayers, with the final aim to directly insert it in the biomimetic membrane has been checked [75]. At the same time, with the final aim to miniaturise the detection area of this sensor, the enzymatic polymer membrane of few micrometers thick needs to be removed. Another monoclonal antibody directly inserted in the biomimetic membrane may play the role of a second anchor to fix oxidase required for ECL reaction (hydrogen peroxide generation). This part of the work is now under investigation.

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Finally, the interfacing of ultrathin LB membranes with a high-performance ECL device opens a new way in the achievement of miniaturised bio-optoelectronic sensors and further developments in macroarray systems by insertion of different biocatalytic elements, using the diversity of the antibody recognition specificity.

Figure 8 Principle of reagentless bio-optoelectronic microsensor based on biomimetic LB membrane. Structure of amphiphilic derivative from Tifeng et al. [74, 75].

Screen-printed microelectrode

17 nm

acetylcholine

choline

H2O2

+diazaquinone

hν

λmax. = 425 nm

AChE

ChOD

17 nm

Biomimetic LB membrane (Neoglycolipid - Antibodies - ChOD/AChE )

O O O O

OC11H23

OC11H23

HN

HN

HN

O

O

O

Amphiphilic luminol derivative

1.5.3 Biomimetic sensors to access biogical membrane functioning

The association of Langmuir–Blodgett biomimetic membranes with high performance optical sensors illustrates the way in which ‘natural’ patterning of the sensitive layer by self-association of biomolecules can be combined with surface functionalisation in the design of miniaturised bio-optoelectronic sensors. Besides, direct interfacing of proteo-lipidic LB membrane with a chemical transducer, allowing both recognition and transduction in a single device may be exploited to access complex functioning of biological membrane. The main reason is the direct access of the local environment (i.e. microenvironment) of the biological element without diffusion of the reactants which gives a statistical and global view of the biological phenomena. Due to the intimate contact of an LB membrane associating enzyme with the transducer, it has been possible few years ago to detect instantaneously very low enzyme activity [59]. The absence of diffusion constraints, related to the small enzyme amount retained on the LB films, gives the opportunity to assess the catalytic properties (intrinsic behaviour) of enzymes associated with LB membranes. The possibility of directly studying through LB sensor technology, phenomena such as recognition and transducing of molecular information, which constitutes the main biological process in natural

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membranes, appears then attractive for further developments of models devoted to the investigations of biological processes in a reconstituted biomimetic situation. More recently, the intimate contact of biomimetic LB membranes associating oriented recognition sites with a miniaturised ECL device (Figure 7), allowed to directly investigate the enzyme kinetics thank to an efficient transduction of the biochemical signal, like in the biological membrane [73].

From a more general standpoint, protein–lipid membranes associated with sensors can be used to study their functional properties. This association corresponds to a biomimetic simulation as close as one could hope to get to one of the main functions of biological membranes, namely, the recognition and transduction of biological signals. The direct contact between biological element and transducer allows a detailed study of its recognition properties and the resulting physicochemical modifications, providing information about the structure–function relations of biological membranes. If the protein is an enzyme, one can investigate its catalytic properties in a heterogeneous medium in a biomimetic lipid environment at the nanoscale. In particular, this type of study is relevant in the field of nanobioscience.

1.6 Trends and perspectives

For several years, self-assembly properties of biomolecules received more and more attention because of their ability to spontaneously organize into nanostructures, which allows mimicking the living cell membranes. Langmuir monolayers are membrane models exploiting the self-association properties of amphipathic lipid molecules at the air/water interface. The main advantage with them is the possibility of obtaining a perfectly ordered state at the water surface and then being able to control this aggregated state by varying the imposed surface pressure. In nanobiotechnology, the interest in developing Langmuir monolayers is double-edged. On the one hand, they can be used to form supported lipid bilayers by transferring the monolayer onto a solid substrate, while on the other hand they are well-suited to the study of lipid/protein interactions or of macromolecules in general.

Langmuir-Blodgett technology is a powerful method to elaborate functionalised biomimetic membranes. Different aspects of the biological membrane, like fluidity or asymmetry can be preserved, but the most promising outcome resides in the possibility to orient functional macromolecules in the bilayer structure. By the way to be directly prepared at the surface of different kinds of solid materials, LB membranes present some real advantages for applications in nanobiotechnology and applied nanobiosciences. A direct association with active surfaces constitutes an attractive opportunity for designing novel nanosensors. The intimate contact between LB membranes and effective transducers, allowing recognition and signal transduction events in a single device is without doubt, a very promising way for the development of biomimetic nanosensors and minute investigations of biological processes at the molecular level.

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2 Biochips and microarrays: nanobiotechnology applications

2.1 Biomolecules electro-grafting The immobilization of the biological molecules is a crucial step in the highly innovative biochip research field since it is directly related to the biosensing performances obtained. To date, even if a wide variety of biochips were developed, [76] no generic procedure has merged, which could be easily applied to both protein and nucleic acid, and more generally to interaction-based biochips. Nowadays, an obvious need exists to set-up a flexible immobilization process, providing strong, stable and accessible binding of the sensing-element, thus leading to sensitive and reproducible biochip performances. Our group have recently [77] presented an immobilization strategy enabling the direct grafting of aryl-diazonium modified non-catalytic proteins (antigens) at the surface of screen-printed graphite electrode (SP) biochips. This approach leads to spatially resolved grafting of proteins onto conducting surfaces. For that purpose, the biomolecules were first modified with aniline derivatives, which were oxidized into aryl-diazonium function prior to the electro-addressing (Figure 9).

Figure 9 Strategy for direct electro-addressing of modified antibody onto SP graphite electrode surface. CMA: 4-CarboxyMethylAniline, DDC: N,N'-dicyclohexyl-carbodiimide.

This technique is based on the particular electrochemical property of aryl-diazonium salts [78]. These molecules could be electro-addressed at the surface of a polarized electrode, leading to the formation of a covalent C-X (X being the electrode material) bond between the aryl group and the electrode (Figure 10). This electrochemical-grafting property of aryl-diazonium derivatives was previously confirmed on a large variety of conductive and semi-conductive material such as carbon, metal, silicon, diamond and recently on ITO electrodes [79-83].

We lately demonstrated, in a proof of concept study, the usefulness of this technique for the electro-addressed immobilization of biomolecules as different as antibody, nucleic acid and enzyme [84]. The potentialities of the electro-addressing chemistry are illustrated through different biosensing architecture – i.e. oligonucleotide based assay,

18

capture immunoassay and sandwich immunoassay – all involving a chemiluminescent detection system using horseradish peroxidase as label (Figure 11).

Figure 10 The aniline derivative electro-addressing mechanism: i) diazotation of the aniline derivative, ii) electro-reduction of diazonium at a conductive electrode surface, iii) covalent grafting to the carbon electrode surface via a C-C bound.

NH2

R

iN

R

N

R

N2

ii+e-

iii

R

The first demonstration of an analytical application of the electro-addressing of aryl-diazonium modified biomolecule is based on oligonucleotide functionalized biochip. To our knowledge, only a few works deals with the direct covalent binding of oligonucleotides on conducting material [82, 85]. Here, a 20mer sequence from a "hot spot" of the exon 8 of the p53 tumor suppressor gene [83] was functionalized with 4-aminobenzylamine (4-ABA), electro-addressed and used as stationary phase probe sequence for hybridization testing of biotinylated target sequence (Figure 11-a). The probe sequence was here functionalized at its 5’-end with 4-ABA to provide an orientated grafting. The probe surface density was estimated using XPS experiment (S.I) and was found to be 3.75 10-13 molecules/cm2. This result compares well with similar XPS experiments for surface coverage determination on gold substrate [86]. Similar studies were systematically performed with 4-carboxyaniline (4-CMA) modified proteins as addressed biomolecules. First, rabbit immunoglobulins (IgG) were used as immobilized antigens and involved in the detection of rheumatoid factor (RF) – i.e. a family of human antibodies largely involved in rheumatoid diseases [87] and which the presence could be characterized by an anti-rabbit IgG activity of the serum. The assay, a capture format (Figure 11-b), is not considered as a sandwich assay since the immobilized rabbit IgGs are not used as active antibodies but as capture antigens. Nevertheless, the success of the assay is determined by the accessibility of the different epitopes of the immobilized IgG toward the numerous paratopes of the polyclonal human sera antibodies [88]. Different human serum samples containing known concentrations of rheumatoid factor were incubated on the rabbit IgG modified biochip surface. A clear correlation between the measured chemiluminescent signal and the RF value in the serum samples was found, allowing the detection of RF in the 5.3-485 IU/ml range with an acceptable accuracy when compared to previous works (standard Auraflex® ELISA test and [88]). For the immobilization technique to be fully demonstrated as useful for biosensing, a second immunochemical application based on a sandwich immunoassay procedure has been demonstrated. It involves the recognition properties of electro-addressed anti-human IgGs and the binding properties of the grafted antibodies are directly implicated in the detection process of the antigen in solution, here a human IgG (Figure 11-c). Thus, every loss of integrity of the immobilized antibodies would have a dramatic effect on the recognition event. In this case, we were able to demonstrate that the immobilized anti-human IgG antibodies could be successfully involved in the recognition of the free target

19

protein, evidencing that the proposed immobilization procedure maintains a convenient structural conformation of the grafted biomolecules, allowing them to be implicated in a recognition process.

Figure 11 Schematic representations of the different proofs of concept using the electro-addressed immobilisation of (a) a probe DNA sequence, (b) an immunoglobulin antigen and (c) an active anti-human antibody.

2.2 Gold nanotexturation for the on-chip chemiluminescent enhancement Our group recently described an original method for the enhancement of chemiluminescent (CL) on-chip detection of protein and oligonucleotide [89]. This enhancement is based on the electro-deposition of a gold nanostructured layer onto a screen-printed (SP) carbon microarray, prior to the immobilization of biomolecules through the diazonium adduct electro-deposition process. Morphological studies of the Au layer (optical and atomic force microscopy) show that the metal film is composed of nanostructured (rms 16.5 nm) 800 nm diameter particles covering the entire graphite surface and yielding a high surface area (Figure 12). Using these modified SP microarrays, enhancement factors of 229 and 126 were obtained for prostate-specific

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antigen (PSA) immuno-detection and p53 oligonucleotide detection, respectively. These enhancements were associated with three different phenomena: an enhancement of the catalyzed chemiluminescent reaction by the gold surface, an increase of the specific surface area for immobilization of the probe biomolecules, and an opposite quenching effect due to the overlapping of the gold absorption and the CL emission peaks. For the free PSA and target oligonucleotide detection, enhanced performances were thus obtained, giving detection limits of 5 ng/mL and 0.1 nM, respectively.

Figure 12 Atomic force microscopy (NT-MDT, tapping mode) images of a) a bare SP electrode and b-d) different magnifications of an Au*SP electrode. The individual 800 nm particle roughness is 16.5 nm (rms).

2.3 Direct polymer modification with biomolecules Since the emergence of the miniaturization concept [90] and the need for improved biomedical devices [91], polymers have become very popular materials for the development of micro and nano fabricated biological systems [92]. The early stage was focused on glass or silicon technology, particularly in the field of lab-on-chip devices or surface patterning, but various polymers (for example PDMS, PMMA, PTFE, PS) [93-95] gained rapid interest because of their availability, their cost and the broad range of chemical reactivity allowing an easy and customisable biomolecules grafting [96]. Following this evolution, PDMS appeared rapidly as a powerful material for rapid prototyping and was used for many different applications among which immunoassays

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[97, 98], microfluidics [99, 100], surface patterning [101, 102] and electrophoresis [103]. Indeed, PDMS exhibits several attractive properties required for the development and prototyping of micro fabricated tools [104] such as low cost, ease to use in standard academic laboratory conditions, good optical transparency, gas permeability, biological compatibility due to its low toxicity and finally very low pattern resolution when moulded on a substrate [105, 106]. However, these advantages are counter balanced by a very high hydrophobicity and a chemical inertness of the polymer which triggered the need for efficient surface modification procedures. Most of the time, a strong oxidative step (UV irradiation, UV/ozone treatment or plasma exposure) [107] is applied to generate a hydrophilic glass like surface allowing well known silanization coupling chemistry with biomolecules [108]. All these steps remain time, energy and chemicals consuming and impart some interesting qualities of the PDMS.

Figure 13 Overview of the “Macromolecules to PDMS transfer” technique highlighting the 4

main steps leading to the achievement of protein spots directly and entrapped at the PDMS interface. SEM image of a proteins microarray at the surface of the PDMS.

In order to overcome this surface modification issue, our group developed in the last three years another approach for the direct PDMS functionalization (Figure 13). The method, now called “Macromolecules to PDMS transfer”, allows direct PDMS surface modification with active proteins [109] or modified DNA [110] for the development of biochips. This procedure relies on the ability of the PDMS polymer to entrap macromolecules at its surface while the polymerization process occurs. Briefly, macromolecules spots are patterned using a piezo arrayer on a 3D mould (glass or Teflon) before being covered with liquid PDMS. Then, curing at high temperature rendered the polymer elastomeric enabling its separation from the mould. Once peeled off, the polymer exhibits active protein spots at the PDMS air interface. The main advantage of this concept is to be able to combine both the macromolecule

22

immobilization, without the need of additional chemicals, and the easy 3D structures achievement according to the initial mould structure. Numerous applications were already described using this method and we are presenting herein a focus on the implementation of the technique showing how different proteins can be successfully immobilized and used for (i) the development of sensitive sandwich immunoassay for C-reactive protein (CRP) detection, (ii) the characterisation of patient sera according to rheumatoid factor (RF) level and finally (iii) demonstrate how the procedure can be used to easily and rapidly produce fibronectin based cell culture arrays (Figure 14).

Figure 14 Schematic representation of the sandwich CRP assay (A), the RF capture assay (B) and the cell culture biochip (C), based on the “Macromolecules to PDMS transfer” procedure.

First, C-reactive protein (CRP) sandwich immunoassay using immobilised monoclonal anti-CRP antibodies was demonstrated for sepsis diagnosis. The preserved integrity of the immobilised monoclonal immunoglobulin permitted the sensitive detection of free CRP in human sera (LOD=12.5µg/L, detection ranging over two decades). Then, rheumatoid arthritis diagnosis through the rheumatoid factor (RF) detection based on rabbit immunoglobulins immobilisation was shown to allow the detection of specific antibodies in human sera samples down to low RF levels (detection range 5.3-485 IU/mL). Finally, the “Macromolecules to PDMS transfer” procedure was used to easily and rapidly produce fibronectin based cell culture arrays. The successful attachment of HeLa and BALB/3T3 cells was demonstrated with optical microscopy and specific staining of actin and vinculin (Figure 15). The extension of such localised cell culture is now under investigation for the characterisation of nanostructure/cells interactions and mechanisms.

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Figure 15 Optical microscope images of HeLA cells cultured onto 150µm fibronectin spots. Phase contrast image (A), FITC fluorescent image after vinculin immuno-staining (B), Alexa Fluor® 546 fluorescent image after filamentous actin staining (C) and DAPI nuclei staining fluorescent image (D).

Acknowledgements This work was partially supported by (i) French minister MENRT (Ministère de l’éducation Nationale de la Recherche et Technologie) for Doctoral fellowship of Miss Stéphanie Godoy-Violot (2001-2004), (ii) CNRS (Centre National de la Recherche Scientifique), department of chemical science, section 12, (N°06-209) for post-doctoral fellowship of Dr. Tifeng Jiao (October 2006 – September 2007) and (ii) Centre de Compétences en nanosciences de la région Rhône-Alpes, C'Nano Rhône-Alpes for Brewster Angle Microscope acquisition.

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